13.4 Human Identity Testing & Post-Transplant Chimerism

13.4 Human Identity Testing & Post-Transplant Chimerism

Quick Summary: Human identity testing, forensic individualization, parentage testing, and post-transplant chimerism analysis exploit hypervariable Short Tandem Repeats (STRs), also known as microsatellites ($2–6\text{ bp}$ repeat units). The FBI Combined DNA Index System (CODIS) standardizes a core panel of 20 autosomal STR loci plus the Amelogenin (AMELX/Y) sex-typing marker. Alleles are amplified in fluorescent multiplex PCR assays and resolved by Capillary Electrophoresis (CE) to within single-base resolution against an allelic ladder. In bone marrow and hematopoietic stem cell transplantation (HSCT), STR markers distinguish recipient from donor hematopoiesis to monitor engraftment, mixed chimerism, and early leukemic relapse.

---

1. Short Tandem Repeats (STRs) & The FBI CODIS Core Loci

Autosomal STR loci are highly polymorphic, non-coding tetranucleotide repeat tracts scattered throughout the human genome that follow Mendelian co-dominant inheritance.

+----------------------------------------------------------------------------------------------------+
|                                 THE 20 CODIS CORE AUTOSOMAL STR LOCI                               |
+-------------------+-------------------+-------------------+----------------------------------------+
| STR Locus Name    | Chromosomal Locus | Repeat Motif      | Allelic Range & Repeat Characteristics |
+-------------------+-------------------+-------------------+----------------------------------------+
| **TH01**          | **11p15.5**       | **[AATG]$_n$**    | 5 to 11 repeats; includes microvariant  |
|                   | (Intron 1 of *TH*)|                   | **9.3** (9 full repeats + 3 bp [ATG])  |
+-------------------+-------------------+-------------------+----------------------------------------+
| **TPOX**          | **2p25.3**        | **[AATG]$_n$**    | 6 to 13 repeats; intron 10 thyroid perox|
+-------------------+-------------------+-------------------+----------------------------------------+
| **CSF1PO**        | **5q33.1**        | **[AGAT]$_n$**    | 6 to 15 repeats; c-fms proto-oncogene  |
+-------------------+-------------------+-------------------+----------------------------------------+
| **vWA**           | **12p13.31**      | **[TCTG][TCTA]$_n$| 11 to 24 repeats; von Willebrand gene  |
+-------------------+-------------------+-------------------+----------------------------------------+
| **D3S1358**       | **3p21.31**       | **[TCTA][TCTG]$_n$| 12 to 19 repeats; complex repeat       |
+-------------------+-------------------+-------------------+----------------------------------------+
| **D8S1179**       | **8q24.13**       | **[TCTA][TCTG]$_n$| 8 to 19 repeats; complex repeat        |
+-------------------+-------------------+-------------------+----------------------------------------+
| **D21S11**        | **21q21.1**       | **Complex tetra** | 24 to 38 repeats; complex microvariants|
+-------------------+-------------------+-------------------+----------------------------------------+
| **FGA**           | **4q31.3**        | **[CTTT]$_n$**    | 17 to 30+ repeats; alpha-fibrinogen    |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Additional 12   | D1S1656, D2S441, D2S1338, D5S818, D7S820, D10S1248, D12S391, D13S317,      |
| CODIS Loci**      | D16S539, D18S51, D19S433, D22S1045; plus optional Penta D, Penta E      |
+-------------------+-------------------+-------------------+----------------------------------------+

Amelogenin Sex Determination Assay

The Amelogenin gene (AMELX on Xp22.2; AMELY on Yp11.2) is not an STR, but a sequence-conserved gene used in identity testing to establish biological sex:

• Structure: Intron 1 of AMELX harbors a 6-bp deletion (positions 106 bp) relative to the homologous region in AMELY (112 bp).
• Females ($XX$): Produce a single fluorescent peak at $106\text{ bp}$ (homozygous $X$).
• Males ($XY$): Produce two fluorescent peaks at $106\text{ bp}$ and $112\text{ bp}$ with an approximately equal $1:1$ peak height ratio.
• Technical Caveat: Rare phenotypic males with a deletion of the AMELY locus on Yp11.2 can type falsely as female (single 106 bp peak); resolved by testing male-specific Y-STR markers (e.g., DYS19, DYS389I/II, DYS390).

                           AMELOGENIN CAPILLARY ELECTROPHEROGRAM
                           
      [ Female Profile (XX) ]                      [ Male Profile (XY) ]
      Fluorescence                                 Fluorescence
         ^
         |        ||                                  |        ||          ||
         |        || (106 bp)                         |        || (106 bp) || (112 bp)
         |        ||                                  |        ||          ||
         +--------+-----------------> Size            +--------+-----------+------> Size

---

2. Technical Artifacts in STR Capillary Electrophoresis

Accurate interpretation of STR electropherograms requires distinguishing true biological alleles from in vitro enzymatic and optical artifacts.

+----------------------------------------------------------------------------------------------------+
|                         TECHNICAL ARTIFACTS IN CAPILLARY ELECTROPHORESIS                           |
+-------------------+-------------------+-------------------+----------------------------------------+
| Artifact Type     | Molecular Cause   | Peak Characteristics| Laboratory Remediation Strategy       |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Stutter**       | DNA polymerase    | Minor peak **one  | Filter using locus-specific stutter    |
|                   | slippage during   | repeat unit shorter| thresholds (typically $\le 15\%$ of     |
|                   | in vitro PCR      | ($-4\text{ bp}$)**| true parent peak height)               |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Split Peaks /   | Incomplete non-   | True amplicon ($N$)| Perform an extended post-PCR terminal  |
| Minus-A (-A)**    | templated adenine | alongside adenyl- | incubation ($60–72^\circ\text{C}$ for  |
|                   | addition by Taq   | ated form ($N+1$),| $30–60\text{ min}$) to force 100% $+A$ |
|                   |                   | $1\text{ bp}$ apart| conversion                             |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Pull-Up /       | Incomplete matrix | Phantom peak at   | Re-run spectral matrix calibration;    |
| Bleed-Through**   | deconvolution;    | identical base-pair| dilute overloaded amplicon to reduce   |
|                   | excessive signal  | size in an adjacent| fluorescent saturation                 |
|                   | (>8,000 RFU)      | dye channel       |                                        |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Allelic         | Stochastic low-   | Complete loss of  | Increase template input ($>0.5–1\text{ ng}$);|
| Drop-Out**        | template sampling | one true allele in| redesign primers if binding site       |
|                   | or primer mutation| a heterozygote    | polymorphism is suspected              |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Microvariants** | Incomplete repeat | Real biological   | Aligns precisely to designated decimal |
| (Off-Ladder)**    | units (e.g., TH01 | allele (e.g., 9.3)| bins in allelic ladder; verified by    |
|                   | 9.3 = 9x + 3 bp)  |                   | re-injection or sequencing             |
+-------------------+-------------------+-------------------+----------------------------------------+

                             ELECTROPHEROGRAM ARTIFACT PROFILES
                             
      [ Stutter Artifact ]         [ Minus-A Split Peak ]        [ Pull-Up / Bleed-Through ]
      Fluorescence                 Fluorescence                  FAM Channel (Blue Overload):
         ^                            ^
         |       || (True Peak)       |       || (N+1 Form)           |     ||||| (>8000 RFU)
         |   |   ||                   |   ||  || (N Form)        VIC Channel (Green Pull-Up):
         |   |   ||                   |   ||  ||                      |
         +---+---+-------->           +---+---+-------->              |       |   (Phantom Peak!)
           N-4   N                      N    N+1                      +-------+--------------->

---

3. Paternity Index & Probability of Paternity Calculations

In parentage testing, genetic markers are evaluated to determine whether an alleged father (AF) can be excluded from biological paternity, or to calculate the statistical weight of inclusion.

Direct Exclusion Criteria

• Direct Exclusion (Class I): The child possesses an obligate paternal allele that is absent in both the biological mother and the alleged father (e.g., Mother: $12, 12$; Child: $12, 14$; Obligate Paternal Allele = $14$; Alleged Father: $10, 11$).
• Exclusion Standard: Modern legal standards require direct exclusions at $\ge 2–3$ independent STR loci to confirm non-paternity, preventing a false exclusion caused by a sporadic single-locus germline mutation.

Statistical Paternity Index Formula

The Paternity Index ($PI$) at an individual locus represents the likelihood ratio comparing the probability of observing the child's genotype if the alleged father is the biological father ($X$) versus if a random unrelated man from the population is the father ($Y$):

$PI = \frac{X}{Y}$

• If the Alleged Father is homozygous for the obligate paternal allele: $X = 1.0$; $PI = \frac{1.0}{p}$
• If the Alleged Father is heterozygous for the obligate paternal allele: $X = 0.5$; $PI = \frac{0.5}{p}$ (where $p$ is the population frequency of the obligate paternal allele).

$\text{Combined Paternity Index (CPI)} = PI_1 \times PI_2 \times PI_3 \times \dots \times PI_n$

$\text{Probability of Paternity } (W) = \frac{CPI \times P_0}{(CPI \times P_0) + (1 - P_0)} \times 100\%$

Assuming a neutral prior probability ($P_0 = 0.5$), the equation simplifies to:

$W = \frac{CPI}{CPI + 1} \times 100\%$

(A $CPI \ge 100$ corresponds to $W \ge 99.0%$, and $CPI \ge 10,000$ achieves legal proof of paternity with $W \ge 99.99%$).

---

4. Post-Allogeneic Stem Cell Transplant Chimerism Monitoring

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy for leukemias, lymphomas, and marrow failure disorders. Post-transplant chimerism testing quantifies the proportion of donor versus recipient cells in the host bone marrow or peripheral blood.

+----------------------------------------------------------------------------------------------------+
|                                 CHIMERISM STATUS DEFINITIONS                                       |
+-------------------+-------------------+------------------------------------------------------------+
| Chimerism State   | % Donor Hematopoiesis| Clinical Interpretation & Management                    |
+-------------------+-------------------+------------------------------------------------------------+
| **Complete / Full | **100% Donor**    | **Successful Engraftment**: Complete eradication of        |
| Chimerism**       | ($<1\%$ Recipient)| recipient marrow; minimal risk of immediate relapse        |
+-------------------+-------------------+------------------------------------------------------------+
| **Mixed           | **1% to 99% Donor**| **Coexistence of Donor & Host**: May be stable (tolerance) |
| Chimerism**       |                   | or progressive (impending graft rejection or relapse)      |
+-------------------+-------------------+------------------------------------------------------------+
| **Split-Lineage / | Variable across   | Evaluates purified fractions: **CD3+ T-cells**,            |
| Lineage-Specific**| T, B, & Myeloid   | **CD19+ B-cells**, and **CD33+/CD15+ Myeloid cells**       |
+-------------------+-------------------+------------------------------------------------------------+
| **Graft Failure / | **< 1% Donor**    | **Rejection / Relapse**: Complete loss of donor cells with |
| Rejection**       | (>99% Recipient)  | autologous host marrow reconstitution or leukemic recurrence|
+-------------------+-------------------+------------------------------------------------------------+

                               INFORMATIVE STR LOCUS PROFILES
                               
      [ Fully Informative Locus ]             [ Partially Informative Locus ]
      Recipient: Alleles 12, 14               Recipient: Alleles 10, 12
      Donor:     Alleles 10, 11               Donor:     Alleles 10, 14
      (All peaks distinct and non-shared!)    (Allele 10 is shared; 12 and 14 are unique)
      
      Fluorescence                            Fluorescence
         ^                                       ^
         |    ||      ||     ||      ||          |    ||          ||          ||
         |    ||      ||     ||      ||          |    || (Shared) || (Recip)  || (Donor)
         +----+-------+------+-------+----->     +----+-----------+-----------+----->
             D:10    D:11   R:12    R:14             10          12          14

---

5. Quantitative Chimerism Calculations & Lineage Sorting

Peak Area Chimerism Formula

For an informative STR locus displaying distinct recipient-specific ($R_1, R_2$) and donor-specific ($D_1, D_2$) peaks:

$\%\text{ Recipient DNA} = \left( \frac{\sum \text{Area}_{\text{Recipient Unique Peaks}}}{\sum \text{Area}_{\text{Recipient Unique Peaks}} + \sum \text{Area}_{\text{Donor Unique Peaks}}} \right) \times 100\%$

$\%\text{ Donor DNA} = 100\% - \%\text{ Recipient DNA}$

+----------------------------------------------------------------------------------------------------+
|                   LINEAGE-SPECIFIC CELL SORTING IN POST-TRANSPLANT HSCT                            |
+-------------------+-------------------+-------------------+----------------------------------------+
| Cell Population   | Surface Marker    | Isolation Method  | Clinical Value & Diagnostic Utility    |
+-------------------+-------------------+-------------------+----------------------------------------+
| **T-Lymphocytes** | **CD3+**          | Magnetic Bead     | Assesses **graft-versus-host / graft-  |
|                   | (Pan T-cell)      | (MACS) or FACS    | versus-leukemia (GvL)** potential; pre-|
|                   |                   |                   | dicts early graft rejection            |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Myeloid Cells** | **CD33+ / CD15+ / | Magnetic Bead     | Detects **early acute myeloid leukemia |
|                   | CD14+**           | (MACS) or FACS    | (AML) relapse**; myeloid cells have    |
|                   |                   |                   | short half-life ($<24\text{ hrs}$)     |
+-------------------+-------------------+-------------------+----------------------------------------+
| **B-Lymphocytes** | **CD19+ / CD20+** | Magnetic Bead     | Monitors B-ALL recurrence and post-    |
|                   |                   | (MACS) or FACS    | transplant lymphoproliferative disorder|
+-------------------+-------------------+-------------------+----------------------------------------+

+----------------------------------------------------------------------------------------------------+
|                         CHIMERISM TESTING PLATFORMS COMPARISON                                     |
+-------------------+-------------------+-------------------+----------------------------------------+
| Methodology       | Limit of Detection| Quantitative Range| Key Strengths & Technical Trade-Offs   |
+-------------------+-------------------+-------------------+----------------------------------------+
| **STR-PCR with    | **1.0% to 5.0%**  | 1% to 100%        | Standard, cost-effective, multiplexed; |
| Capillary Elect.**|                   |                   | lacks sensitivity for deep MRD         |
+-------------------+-------------------+-------------------+----------------------------------------+
| **Droplet Digital | **0.01% to 0.1%** | 0.01% to 100%     | Absolute Poisson quantification;       |
| PCR (ddPCR)**     |                   |                   | highly sensitive for early host relapse|
+-------------------+-------------------+-------------------+----------------------------------------+
| **Targeted NGS    | **0.05% to 0.1%** | 0.05% to 100%     | Simultaneous profiling of hundreds of  |
| SNP Panels**      |                   |                   | informative SNPs; high throughput      |
+-------------------+-------------------+-------------------+----------------------------------------+